Immobilised biological entities

ABSTRACT

There is described inter alia a medical device having a surface which comprises a coating layer, said coating layer being a biocompatible composition comprising an entity capable of interacting with mammalian blood to prevent coagulation or thrombus formation, which entity is covalently attached to said surface through a link comprising a 1,2,3-triazole.

This invention relates to immobilised biological entities, surfaces,e.g. of medical devices, coated with such entities, and processes andintermediates for their production.

BACKGROUND OF THE INVENTION

When a medical device is placed in the body, or in contact with bodyfluids, a number of different reactions are set into motion, some ofthem resulting in the coagulation of the blood in contact with thedevice surface. In order to counteract this serious adverse effect, thewell-known anti-coagulant compound heparin has for a long time beenadministered systemically to patients before the medical device isplaced in their body, or when it is in contact with their body fluids,in order to provide an antithrombotic effect.

Thrombin is one of several coagulation factors, all of which worktogether to result in the formation of thrombi at a surface in contactwith the blood. Antithrombin (also known as antithrombin III) (“AT”) isthe most prominent coagulation inhibitor. It neutralizes the action ofthrombin and other coagulation factors and thus restricts or limitsblood coagulation. Heparin dramatically enhances the rate at whichantithrombin inhibits coagulation factors.

However, systemic treatment with high doses of heparin is oftenassociated with serious side-effects of which bleeding is thepredominant. Another rare, but serious complication of heparin therapyis the development of an allergic response called heparin inducedthrombocytopenia that may lead to thrombosis (both venous and arterial).High dose systemic heparin treatment e.g. during surgery also requiresfrequent monitoring of the activated clotting time (used to monitor andguide heparin therapy) and the corresponding dose adjustments asnecessary.

Therefore solutions have been sought where the need for a systemicheparinisation of the patient would be unnecessary or can be limited. Itwas thought that this could be achieved through a surface modificationof the medical devices using the anti-coagulative properties of heparin.Thus a number of more or less successful technologies have beendeveloped where a layer of heparin is attached to the surface of themedical device seeking thereby to render the surface non-thrombogenic.For devices where long term bioactivity is required, heparin shoulddesirably be resistant to leaching and degradation.

Heparin is a polysaccharide carrying negatively charged sulfate andcarboxylic acid groups on the saccharide units. Ionic binding of heparinto polycationic surfaces was thus attempted, but these surfacemodifications suffered from lack of stability resulting in lack offunction, as the heparin leached from the surface.

Thereafter different surface modifications have been prepared whereinthe heparin has been covalently bound to groups on the surface.

PRIOR ART

One of the most successful processes for rendering a medical devicenon-thrombogenic has been the covalent binding of a heparin fragment toa modified surface of the device. The general method and improvementsthereof are described in European patents: EP-B-0086186, EP-B-0086187,EP-B-0495820 and U.S. Pat. No. 6,461,665.

These patents describe the preparation of surface modified substrates byfirst, a selective cleavage of the heparin polysaccharide chain, e.g.using nitrous acid degradation, leading to the formation of terminalaldehyde groups. Secondly, the introduction of one or more surfacemodifying layers carrying primary amino groups on the surface of themedical device, and thereafter reacting the aldehyde groups on thepolysaccharide chain with the amino groups on the surface modifyinglayers followed by a reduction of the intermediate Schiff's bases toform stable secondary amine bonds.

However there is still a requirement for surface modifications which aremore easily manipulated, are simpler and more efficient to produceand/or where the bioavailability of the heparin moiety is higher.

Baskin et al QSAR Comb. Sci. 26, 2007, No. 11-12, 1211-1219 describe theuse of the reaction of azides with alkynes for the covalent labeling ofbiomolecules in cells and living organisms, but play down its usebecause of the toxic nature of the copper used to catalyze the reaction.

US patent applications 20070020620, 20050032081 and 20050222427 relateto the use of a similar reaction to attach biomolecules to various othermolecules.

WO2007/003054 (Shoichet) discloses the immobilization of biomolecules onpolymers. Specifically, biodegradable polymers are mentioned (page 1,line 13). Reaction of an alkyne with an azide to form a triazole isillustrated. However application to the preparation of compositionshaving anti-coagulant function is not envisaged. Moreover use of atriazole to achieve linkage of a biomolecule to the surface of anymedical device is not envisaged either.

EP1806373 (Cordis) describes a neutral tri-branched polymer for coatingmedical devices. The polymer is typically dip or spray-coated onto thedevice which is not pre-treated in any way. The preferred heparin to beemployed is low molecular weight (i.e. degraded) heparin. The disclosureappears speculative and although a process for attachment of heparin tothe polymer scaffold is shown in Scheme 1, the suggested product (shownas structure III) seems unlikely to be produced since the NHS moiety ofthe molecule that is reacted with heparin should be displaced by aprimary amino group not a hydroxyl group. Heparin contains very fewprimary amino groups, unless these are generated through chemicalprocessing, and none is located at the end-point of the molecule.

US2009/0018646 (Zhao), published after the claimed priority date of thisapplication, describes a biodegradable or bioabsorbable neutral polymerhaving heparin moieties linked thereto via a 1,2,3-triazole. The polymeris typically dip or spray-coated onto the device which may bear a firstdrug-bearing polymer layer but otherwise is not pre-treated in any way.The heparin employed is typically either low molecular weight heparin(see claim 3) or desulfated heparin (see claim 4) to reveal amino groups(not being at the end point of the molecule) which are used as the pointof attachment to the polymer. This reaction is not practically suitablefor use with native heparin which contains hardly any primary aminogroups.

We have now found a simple method of covalently attaching entitiescapable of interacting with mammalian blood to prevent coagulation orthrombus formation, e.g. heparin, and especially full length heparinrather than the degraded heparin of the prior art, to a surface.

SUMMARY OF THE INVENTION

According to the invention we provide, inter alia, a medical devicehaving a surface which comprises a coating layer, said coating layerbeing a biocompatible composition comprising an entity capable ofinteracting with mammalian blood to prevent coagulation or thrombusformation (herein “entity” or “entities” or “anti-coagulant entity” or“anti-coagulant entities”), which entity is covalently attached to saidsurface through a link comprising a 1,2,3-triazole.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows photographs of examples of PVC tubing where the luminalside is coated with nitrous acid degraded heparin or native heparinunder various conditions according to the invention as described inExample 1.5a.

FIG. 2 shows photographs of examples of PVC tubing where the luminalside is processed as described in Example 1.5b and illustrating thatthese tubes are not properly coated with nitrous acid degraded heparin.

FIG. 3 shows photographs of examples of various different substratescoated with nitrous acid degraded heparin according to the invention asdescribed in Example 1.6.

DETAILED DESCRIPTION OF THE INVENTION

Such entities are well known to those skilled in the art and many ofthem are oligosaccharides or polysaccharides. Some of the entities areglycosaminoglycans including compounds containing glucosamine,galactosamine, and/or uronic acid. Preferred glycosaminoglycans are“heparin moieties” and especially full length heparin (i.e. nativeheparin).

The term “heparin moiety” refers to a heparin molecule, a fragment ofthe heparin molecule, or a derivative or analogue of heparin. Heparinderivatives can be any functional or structural variation of heparin.Representative variations include alkali metal or alkaline earth metalsalts of heparin, such as sodium heparin (e.g., Hepsal or Pularin),potassium heparin (e.g., Clarin), lithium heparin, calcium heparin(e.g., Calciparine), magnesium heparin (e.g., Cutheparine), and lowmolecular weight heparin (prepared by e.g. oxidative depolymerization ordeaminative cleavage, e.g. Ardeparin sodium or Dalteparin). Otherexamples include heparan sulfate, heparinoids, heparin based compoundsand heparin having a hydrophobic counter-ion. Other desirable entitiesinclude synthetic heparin compositions referred to as “fondaparinux”compositions involving antithrombin III-mediated inhibition of factorXa. Additional derivatives of heparin include heparins and heparinmoieties modified by means of e.g. periodate oxidation (U.S. Pat. No.6,653,457) and other modification reactions know in the art. Heparinmoieties also include such moieties bound to a linker or spacer asdescribed below. De-sulphated heparin is less preferred because of itsreduced bioactivity relative to other forms of heparin.

We prefer the heparin moiety to be single point attached. We prefer thesingle point to be end point attached. We also prefer the end pointattached heparin to be connected through its reducing end (sometimesreferred to herein as position C1 of the reducing terminal). Theadvantage of end point attachment, especially reducing end pointattachment, is that it is expected that the biological activity of theheparin moiety is maximized due to enhanced availability of the thrombininteraction sites as compared with attachment elsewhere in the heparinmoiety.

Where there are a multiplicity of entities e.g. heparin moieties it ispossible for some or all of them to be of a different type; however weprefer them all to be of the same type.

At its simplest the link consists of the triazole ring only. Howevermore usually the triazole ring will be separated by a spacer from eitherthe surface or the heparin moiety or both. The Mw (molecular weight) ofthe link is suitably from 10² to 10⁶ Da . The length of the link issuitably from 10 to 10³ Å. We prefer the links and/or spacers to bestraight chain(s). It is also possible (although less preferred) forseveral, i.e. more than one, e.g. from 2 to 100, preferably 30 to 100entities (e.g. heparin moieties) to be attached to a single link thusproducing a branched link in which there are several heparin moiety sidechains. In some embodiments the linker includes one or more aromaticrings. In other embodiments the linker does not include any aromaticrings except the triazole ring. In some embodiments the linker ishydrophilic, for example, it may comprise a PEG chain. In one aspect,the link may be viewed as having three portions—“link A” between thesurface and the triazole moiety, the triazole moiety, and “link B”between the triazole moiety and the entity. In one embodiment themolecular weight of link A is between 10¹ and 10³ Da. In anotherembodiment the molecular weight of link B is between 10¹ and 10³ Da. Inone embodiment link A comprises one or more aromatic rings. In anotherembodiment link A does not comprise any aromatic rings. In oneembodiment link B comprises one or more aromatic rings. In anotherembodiment link B does not comprise any aromatic rings. In oneembodiment link A is hydrophilic. In another embodiment link B ishydrophilic. In one embodiment link A comprises a PEG chain. In anotherembodiment link B comprises a PEG chain. In one embodiment links A and Bare both hydrophilic, for example they each comprise a PEG chain. Asused herein, a PEG chain refers to a polymeric chain obtainable bypolymerisation of ethylene oxide, typically of weight between 10² and10⁶ Da. In another aspect, the link may comprise two or more triazolerings. For example, as described in the Examples, use of a bifunctionallinker moiety (such as a bis-azide) can be connected at each end,respectively, to an alkyne functionalized entity and an alkynefunctionalized surface resulting in the link containing two triazolerings. Alternatively, use of a bis-alkyne linker can be connected ateach end, respectively, to an azide functionalized entity and an azidefunctionalized surface also resulting in the link containing twotriazole rings. Thus in another embodiment, the link may be viewed ashaving five portions—“link A” between the surface and a first triazolemoiety, the first triazole moiety, “link B” between the first triazolemoiety and a second triazole moiety, the second triazole moiety, and“link C” between the triazole moiety and the entity. In one embodimentthe molecular weight of link A is between 10¹ and 10³ Da. In oneembodiment the molecular weight of link B is between 10² and 10⁶ Da. Inone embodiment the molecular weight of link C is between 10¹ and 10³ Da.In one embodiment link A and/or link B and/or link C is hydrophilic forexample comprising a PEG chain. For example link B (at least) maycomprise a PEG chain.

Thus suitably the link between the anti-coagulant entity such as aheparin moiety and the surface is an unbranched link and specificallydoes not include a branch of a hydrophobic or hydrophilic polymericmoiety. If a branch is to be included suitably it is only a branchcontaining another anti-coagulant entity such as a heparin moiety.

The link can be biodegradable or non-biodegradable. We prefer the linkto be non-biodegradable in order that a coated medical device isnon-thrombogenic for a long period of time.

Where there is a multiplicity of links it is possible for some or all ofthem to be of a different type; however we prefer all the links to be ofthe same type.

The surface may comprise a coating layer on a solid object, e.g. ashaped object such as a device, and more particularly a medical device.The solid object may have one or more portions containing void spaces,or pores. The pores may be within the object and/or comprising at leastone surface of the object. An example of a porous solid object isexpanded polytetrafluoroethylene (ePTFE).

The solid object may carry one or more, e.g. 2 or more, or 3 or 4 or 5e.g. up to 20 coating layers such that desirably a portion of thesurface (desired to be nonthrombogenic) or the whole of the surface ofthe object is covered (Multilayer Thin Films ISBN: 978-3-527-30440-0).The optimum number of layers will depend on the type of material fromwhich the solid object is made, and the contemplated use of the surface.The surface may, if desired, be made up layer by layer. The number andnature of the layers needed to provide a full coverage of the surfacecan be easily determined by those skilled in the art. The coatinglayer(s) may be formed by adsorbing on the surface of the solid object ahigh average molecular weight cationic polymer, e.g. a polyamine (e.g.that known as Polymin available from BASF, see also EP 0086187 Larssonand Wander) and if needed cross-linking the polyamine with, e.g. analdehyde crosslinker such as crotonaldehyde and/or glutaraldehyde,followed by the application of a solution of an anionic polymer, e.g. ananionic polysaccharide, e.g. dextran sulfate, to obtain at least oneadsorbed layer of the polysaccharide. Hence the surface may comprise alayer of high average molecular weight polyamine and a layer of anionicpolysaccharide. More generally, the surface may comprise one or morecoating bilayers of cationic polymer (e.g. polyamine) and anionicpolymer (e.g. anionic polysaccharide), the innermost layer being a layerof cationic polymer and the outermost layer being a layer of cationicpolymer covalently attached to the entity. This coating procedure isperformed essentially as described in EP-B-0495820. Thus it is only theouter coating layer which is attached to the entity. Typically the outercoating later which is attached to the entity is not cross-linked.

The procedure of EP-B-0495820 may however be modified so that the outerlayer is the anionic polysaccharide which is then reacted, as describedbelow, with a polyamine to which the entity or an azide or alkyne isattached.

Prior to applying the first coating layer the surface of the solidobject, e.g. the medical device, may be cleaned to improve adhesion andsurface coverage. Suitable cleaning agents include solvents as ethanolor isopropanol (IPA), solutions with high pH like solutions comprising amixture of an alcohol and an aqueous solution of a hydroxide compound(e.g. sodium hydroxide), sodium hydroxide solution as such, solutionscontaining tetramethyl ammonium hydroxide (TMAH), acidic solutions likePiranha (a mixture of sulfuric acid and hydrogen peroxide), and otheroxidizing agents including combinations of sulfuric acid and potassiumpermanganate or different types of peroxysulfuric acid orperoxydisulfuric acid solutions (also as ammonium, sodium, and potassiumsalts).

Thus an aspect of the invention is a medical device having a surfacewherein the surface comprises one or more coating bilayers of cationicpolymer and anionic polymer, the innermost layer being a layer ofcationic polymer and the outermost layer being a layer of cationicpolymer covalently attached to the entity.

Another aspect of the invention is a non-thrombogenic medical devicehaving a surface comprising a functionalized cationic polymer outerlayer whereby an anti-coagulant entity is attached to the cationicpolymer outer layer by means of a link comprising a 1,2,3-triazole.

Another aspect of the invention is a non-thrombogenic medical devicewhich is obtainable by a process comprising:

-   -   (a) treating a medical device to present a cationic polymer        surface layer which has been functionalized to bear azido        groups;    -   (b) reacting said cationic polymer surface layer which has been        functionalized to bear azido groups with an anti-coagulant        entity which is functionalized to bear an alkyne group;    -   thereby to attach the anti-coagulant entity to the device        through a link comprising a 1,2,3-triazole.

Another aspect of the invention is a non-thrombogenic medical devicewhich is obtainable by a process comprising:

-   -   (a) treating a medical device to present a cationic polymer        surface layer which has been functionalized to bear alkyne        groups;    -   (b) reacting said cationic polymer surface layer which has been        functionalized to bear alkyne groups with an anti-coagulant        entity which is functionalized to bear an azido group;    -   thereby to attach the anti-coagulant entity to the device        through a link comprising a 1,2,3-triazole.

Another aspect of the invention is a non-thrombogenic medical devicewhich is obtainable by a process comprising:

-   -   (a) treating a medical device to present a cationic polymer        surface layer;    -   (b) associating with said cationic polymer surface layer a        functionalized cationic polymer bearing a plurality of        negatively charged anti-coagulant entities such as heparin        moieties which are attached thereto via a link comprising a        1,2,3-triazole said cationic polymer bearing a plurality of        negatively charged anti-coagulant entities having a net negative        charge.

As described above, the cationic polymer surface may be prepared bytreating the device with a high average molecule weight cationic polymersuch as a polyamine and if necessary cross-linking it with e.g. analdehyde cross-linker. Further layers may optionally be built up bysuccessive steps of (i) application of a solution of anionic polymer(e.g. anionic polysaccharide) to obtain an absorbed layer of the anionicpolymer and (ii) then further treating that with functionalized cationicpolymer, such as a polyamine, to provide an absorbed outer layer offunctionalized cationic polymer, the outermost layer beingfunctionalized to bear azido groups or alkyne groups.

Typically the first step of treating the device with a high averagemolecule weight cationic polymer is preceded by the step of cleaning thesurface of the device with suitable cleaning agents (e.g. thosementioned above) or other methods of surface pretreatment known in theart to improve adherence and coverage of the first layer e.g. polyaminelayer.

Another aspect of the invention is a non-thrombogenic medical devicewhich is obtainable by a process comprising:

-   -   (a) treating a medical device to present an anionic polymer        surface layer;    -   (b) associating with said anionic polymer surface layer a        functionalized cationic polymer bearing a plurality of        negatively charged anti-coagulant entities such as a heparin        moieties which are attached thereto via a link comprising a        1,2,3-triazole said functionalized cationic polymer bearing a        plurality of negatively charged anti-coagulant entities having a        net positive charge.

As described above, the device which presents an anionic polymer surfacelayer is typically prepared by treating the device with a high averagemolecule weight cationic polymer, such as a polyamine, optionally withcross-linking, followed by treating the polyamine surface with asolution of anionic polymer (e.g. anionic polysaccharide) to obtain anabsorbed outer layer of the anionic polymer. Further layers may be builtup by successive steps of (i) application of a cationic polymer(optionally with cross-linking) to provide an absorbed layer of cationicpolymer and (ii) then treating that with a solution of anionic polymer(e.g. anionic polysaccharide) to obtain an absorbed outer layer of theanionic polymer.

Suitably the anionic polymer is a polysaccharide such as dextran sulfateor a derivative thereof.

As used herein a “polyamine” is a molecule having multiple (e.g. 10,100, 1000 or more) free pendant amino groups preferably containing atleast some primary amino groups. Polyamines are typically polymericmolecules having multiple amino groups of high average molecular weight,for example having an average molecular weight of 10³-10⁶ Da. Anexemplary polyamine is a polyethyleneimine such as that known as Polyminavailable from BASF.

The cationic polymer may be functionalized using techniques known in theart. As illustrated in the Examples below, primary amino groups on thepolyamine may be used as points of attachment for the alkyne or azidogroup. However a skilled person would know how to adapt the chemistry touse secondary amino groups on the polyamine as points of attachment forthe alkyne or azido group. Hence polyamines may be functionalized tobear alkyne or azido groups by conventional means e.g. by reactingpendant primary amino groups on the polyamine with an activatedcarboxylic acid (e.g. an N-hydroxy succinimide derivative of acarboxylic acid) which acid bears an alkyne or azido group. Another wayis to react secondary amines with carboxylic acids with carbodiimidechemistry or to react with carboxylic acid chlorides where thecarboxylic acid portion bears an alkyne or azido group.

The entity, e.g. heparin, carrying an alkyne or azido group may be madeby conventional methods known per se. For example an entity, e.g.heparin, carrying an alkyne group may be made by the reaction of analkoxyamine (i.e. molecule of formula R—O—NH₂) carrying an alkyne orazido group with an aldehyde or hemi-acetal group on the entity usingconventional techniques known per se or by methods analogous to thosegiven in the Examples. The connection is formed via an oxy-iminefunction (R—O—N═R′ in which R′ is heparin). Nitrous acid degradedheparin bears an aldehyde group and native heparin contains ahemi-acetal function at their reducing end which may be linked in thisway. The entity, e.g. heparin, carrying an azido group may also be madeby reacting the alkyne functional heparin moiety with an excess of adifunctional azide (e.g. a PEG diazide). A person skilled in the artwill be able to design other ways of introducing an azide or an alkynefunctional group to the reducing end of a carbohydrate chain.

When a coating layer is used, the surface of all and any solid objectsis transformed to present the same functionalized outer surface for thesubsequent attachment of an entity capable of interacting with mammalianblood to prevent coagulation or thrombus formation. Hence a specificadvantage of the processes described herein is that generally a veryuniform non-thrombogenic surface is created (see FIGS. 1 and 3).

The solid object may be, for example a synthetic or naturally occurringorganic or inorganic polymer or material such as polyethylene,polypropylene, polyacrylate, polycarbonate, polyamide, polyurethane(PU), polyvinylchloride (PVC), polyetherketone (PEEK), cellulose,silicone or rubber (polyisoprene), plastics materials, metals, glass,ceramics and other known medical materials or a combination of suchmaterials. Other suitable substrate materials include fluoropolymers,e.g expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene(PTFE), fluorinated ethylene-propylene (FEP), perfluorocarboncopolymers, e.g. tetrafluoroethylene perfluoroalkylvinyl ether(TFE/PAVE) copolymers, copolymers of tetrafluoroethylene (TFE) andperfluoromethyl vinyl ether (PMVE), and combinations of the above withand without crosslinking between the polymer chains.

Suitable metals include nickel titanium alloy (Nitinol), stainlesssteel, titanium, cobalt chromium, gold and platinum. Nitinol andstainless steel are preferred. Titanium is also preferred.

The solid object is suitably a medical device. The medical device can beimplantable or non-implantable. Examples of implantable ornon-implantable medical devices include catheters, stents, stent-grafts,artificial blood vessels, blood indwelling monitoring devices,artificial heart valves, pacemaker electrodes, guidewires,cardiopulmonary bypass circuits, cannulae, balloons, tissue patchdevices, blood pumps, and extracorporeal devices, e.g. extracorporealblood treatment devices, and transfusion devices.

We prefer the coated surface to which the entity (e.g. heparin or otherheparin moiety) is attached to be such that it retains non-thrombogenicproperties after sterilization, e.g. ethylene oxide (EO) sterilization.

Sterilization may be carried out by means well known to those skilled inthe art. The preferred method of sterilization is using ethylene oxidegas. Alternatively, other methods such as radiation, e.g. e-beam orgamma radiation, may be used where such radiation will not degrade theobject or the coating or both.

A preferred embodiment of the present invention relates to a coatedmedical device for implantation e.g. permanent implantation, or otherplacement, at an anatomical site. Other preferred embodiments includetemporary use devices such as catheters and extracorporeal circuits.Examples are sterile (e.g. sterilized) medical devices for placementinside an anatomical structure delimiting a void space, or lumen, toreinforce the anatomical structure or maintain the void space. Suitablythe attached entity, e.g. heparin or other heparin moiety, does notelute to any substantial extent and remains with the device. Forexample, the retained AT binding activity remains adequate (e.g. greaterthan 2 or 4 or 5 or 10 pmol/cm²) and when tested in the Blood loopevaluation test (see Example 1.5a) with 15 hour NaCl (0.15 M) rinseprior to testing with fresh blood from a healthy donor the reduction inplatelet count of the blood after the test is substantially lower forthe blood exposed to the coated surface than that of an uncoated control(e.g. the reduction in platelet count after the test for the bloodexposed to the coated surface is less than 20%, preferably less than 15%and more preferably less than 10%).

Suitably the biocompatible composition of the invention is notbiodegradable or bioabsorbable. For biodegradable or bioabsorbablecompositions the non-thrombogenic properties may generally be expectedto be limited in time.

The non-thrombogenic character of devices according to the presentinvention may be tested by a number of methods. For examplenon-thrombogenic character may be associated with having a highantithrombin binding activity, especially as compared with deviceshaving untreated surfaces.

For example, we prefer the surface, e.g. of the medical device, to havean antithrombin (AT) binding activity of at least 2 e.g. at least 5picomoles AT per square centimeter (pmol/cm²) of surface. In otherembodiments, the AT binding activity is at least 6 pmol/cm², at least 7pmol/cm², at least 8 pmol/cm², at least 9 pmol/cm², or at least 10pmol/cm² of surface. In some embodiments, the AT binding activity is atleast 100 pmol/cm² of surface. AT binding activity can be measured bymethods known in the art, e.g. those described in Pasche., et al., in“Binding of antithrombin to immobilized heparin under varying flowconditions” Artif.—Organs 15:481-491 (1991) and US 2007/0264308. By wayof comparison it may be concluded from Sanchez et al (1997) J. Biomed.Mater. Res. 37(1) 37-42, see FIG. 1, that AT binding values of around2.7-4.8 pmol/cm² (depending on the experimental set up) or more do notappear to give rise to significant thrombogenic enzymatic activity uponcontact with plasma.

Alternatively or additionally we prefer the surface to benon-thrombogenic due to high capacity to suppress coagulation and otherdefence systems in the Blood loop evaluation test described in Example1.5a. According to that test, the surface to be investigated is appliedto a PVC tubing which is rinsed for 15 hours with 0.15M NaCl prior totesting with fresh blood. Non-thrombogenicity is indicated by areduction in platelet count of the blood measured after the test whichis substantially lower for the blood exposed to the surface preparedaccording the method described herein than that of an uncoated control(e.g. the reduction in platelet count after the test for the bloodexposed to the coated surface is less than 20%, preferably less than 15%and more preferably less than 10%).

Other similar blood evaluation methods different from the Blood loopmodel can be performed by those skilled in the art in order to assessthrombogenicity/non-thrombogenicity.

The amount of the entity bound to a particular surface area can becontrolled and adjusted, e.g. by adjusting the amount of the reagentsused in the synthesis of the composition.

The distribution of the entity on the surface can be determined byconventional staining techniques which are known per se, e.g. thedistribution of heparin can be determined using toluidine blue.

According to the invention we also provide a process for the productionof an entity capable of interacting with mammalian blood to preventcoagulation or thrombus formation, which entity is covalently bound to asurface through a link comprising a 1,2,3-triazole, which processcomprises the reaction of a corresponding entity carrying an alkynegroup with a corresponding surface carrying an azido group, or thereaction of a corresponding entity carrying an azido group with acorresponding surface carrying an alkyne group.

This process may be carried out using procedures known per se.

The surface carrying an azido group or an alkyne group may be made byconventional methods known per se, e.g. by reacting a surface, e.g. asurface as described in EP-B-0086186 or EP-B-0086187 carrying negativelycharged sulfate groups with an appropriate polyamine carrying either anazide or an alkyne group respectively.

According to the invention we also provide a polyamine carrying anentity through a link comprising a 1,2,3-triazole.

In one embodiment in which the reaction is used the surface carries theazido group. In another embodiment in which the reaction is used theentity carries the azido group.

The reaction may be carried out in the presence of a metal catalyst, forexample a copper, e.g. a Cu(I) catalyst using reaction conditionsconventionally used in the Huisgen cycloaddition (the 1,3-dipolarcycloaddition of an azide and a terminal alkyne to form a1,2,3-triazole). The Cu(I) catalyst may, if desired, be produced insitu, e.g. by reduction of a corresponding Cu(II) compound for exampleusing sodium ascorbate. The reaction may also, if desired, be carriedout under flow conditions.

As noted in the Prior Art section in connection with the Baskindisclosure, others have commented on the possible toxic nature of theCu(I) catalyst used to catalyze this reaction. However as shown inExample 1.8, our coating appears to be non-toxic. Without being limitedby theory, it is possible that either any residual Cu(I) catalyst iswashed away from the surface or else the polyamine surface complexes itthus rendering it unable to exert any toxic effect.

The reaction may, for example be carried out at a temperature of fromabout 5 to 80° C., preferably at about room temperature. The pH used inthe reaction may be from about 2-12, preferably about 4-9 and mostpreferably at about 7. Suitable solvents include those in which theentity attached to the azide or alkyne is soluble, e.gdimethylsulfoxide, dimethylformamide, tetrahydrofuran and preferablywater or mixtures of water with one of the above. The proportion of theentity to the surface may be adjusted to provide the desired density ofthe entity on the surface. We prefer to use a proportion of the reagentssuch that no free azide or alkyne groups remain on the resultingsurface.

By this new method the entity, e.g. heparin, can advantageously be boundto the surface by surface groups that are not involved in the build upof the surface covering. By contrast, the prior art described inEP-B-0086186, EP-B-0086187 and EP-B-0495820 uses the same type of groups(primary amines) in the layer by layer surface coating process as thoseused to bind the heparin to the coating.

This new process tends to be less sensitive to pH than are the prior artprocesses which is also advantageous.

According to the invention we also provide an entity, e.g. heparin orother heparin moiety, which entity carries an alkyne or an azido group.We also provide a heparin moiety capable of interacting with mammalianblood to prevent coagulation or thrombus formation which entity carriesan alkyne or an azido group, which alkyne or azido group is attached toa linker, wherein the linker is end-point attached to the heparinmoiety. The heparin moiety is suitably a full length heparin moiety(i.e. native heparin).

According to the invention we also provide a functionalized polyaminesurface, e.g. a surface prepared essentially as described inEP-B-0086186, EP-B-0086187 and, EP-B-0495820, but additionally carryingone or more azide or one or more alkyne groups on the outermost layer ofpolyamine.

According to the invention we also provide a medical device having apolyamine surface carrying an azide or an alkyne group e.g. an azide oralkyne group which is connected to an amino group of the polyaminesurface via a link.

According to a further feature of the invention we also provide aprocess for the production of an entity capable of interacting withmammalian blood to prevent coagulation or thrombus formation, whichentity is covalently bound to a surface through a link comprising a1,2,3-triazole, wherein the surface comprises one or more layers ofpolysaccharide (i.e. anionic polysaccharide) and polyamine, whichprocess comprises the reaction of a corresponding surface having anouter layer of polysaccharide (i.e. anionic polysaccharide e.g. carryingnegatively charged sulfate groups) with a polyamine carrying acorresponding entity through a link comprising a 1,2,3-triazole, or thereaction of a corresponding surface having an outer layer ofpolysaccharide (i.e. anionic polysaccharide e.g. carrying negativelycharged sulfate groups) with a polyamine carrying an azide or alkynegroup and reacting the resulting product with an entity carrying analkyne or azido group respectively.

This process for putting down the layers of polysaccharide and polyaminemay be carried out using procedures known per se, for example proceduresanalogous to those described in EP-B-0495820.

According to the invention we also provide a functionalized polyamine,e.g. Polymin which carries one or more azide or one or more alkynegroups e.g. via a linker.

According to the invention we also provide a functionalized polyaminecarrying an entity attached thereto through a link comprising a1,2,3-triazole. This polyamine may be made by procedures known per se,e.g. analogous to those described elsewhere in this specification.

The products of the invention may have one or more of the followingadvantageous properties:

-   -   The degree of substitution of the entity on the surface can be        controlled;    -   Both end-point (one point) attachment and multi-point attachment        of the entity, e.g. heparin, can be achieved, although end point        (especially reducing end point) attachment is preferred;    -   The linker length between the entity and the surface can be        controlled;    -   Full length heparin can be used thus avoiding the cleavage of        heparin and the waste of parts of the cleaved product involved        in the prior art nitrous acid degradation of heparin;    -   When cleaving heparin, the antithrombin binding sequence can be        destroyed in some of the fragments, therefore using full-length        heparin or heparin linked via a spacer can also improve the        bioavailability of the bound heparin;    -   A uniform distribution of the entity over the surface can be        obtained;    -   The bioavailability of the entity can be controlled, e.g. by the        use of different links (length, type);    -   A non-thrombogenic surface which does not leach heparin and        therefore has long lifetime can be obtained.

Other aspects of the invention include a biocompatible compositioncomprising an entity capable of interacting with mammalian blood toprevent coagulation or thrombus formation which entity is covalentlyattached to a surface through a link comprising a 1,2,3-triazole.

The skilled person will appreciate that the biocompatible compositionmay be applied to any solid object, of which a medical device is justone example. Therefore according to another aspect of the inventionthere is provided a solid object having a surface comprising (e.g.coated with) such a biocompatible composition.

The invention is illustrated, but in no way limited, by the followingExamples:

EXAMPLE 1.1 Preparation of a Non-Thrombogenic Surface on Gold

A surface comprising layers of aminated polymer and sulfatedpolysaccharide having a functionalized aminated polymer outer layer isconnected to functionalized heparin thereby forming a triazole ring.

A gold surface (on a quartz crystal microbalance (QCM) crystal) waspretreated using the method described by Larm et al in EP-B-0086186 andEP-495820 (layer-by-layer; polyelectrolyte charge interactions) endingwith a layer of sulfated polysaccharide.

The gold surfaces was first cleaned with ethanol. The priming wasbuilt-up by alternated adsorption of a positively charged polyamine(Polymin) and negatively charged sulfated polysaccharide (dextransulfate). The polyamine is crosslinked with a difunctional aldehyde(crotonaldehyde). Every pair of polyamine and sulfated polysaccharide iscalled one bilayer. The gold surface was primed with 3 bilayers endingwith the sulfated polysaccharide.

Polymin SN (Lupasol SN; Lupasol is an alternative trade name forPolymin) was diluted with water to prepare a stock solution (5 g PolyminSN was added to 20 mL purified water). (Polymin is a polyethyleneiminecationic tenside available from BASF).

The complete process was carried out at a flow of 500 μL/min. in aQ-Sense E4 (http://www.q-sense.se/) system with a peristaltic pump(Ismatec IPC-N 4).

100 μL of a 5% solution of azide functionalized polyamine (preparationsee Example 2a) was added to 100 mL of a 0.04 M/0.04 M borate/phosphatebuffer at pH 8.0. The adsorption of the azide functional polyamine tothe sulfate surface was carried out for 10 minutes at room temperature.A two minute water rinse was performed after the adsorption to rinse offexcess polymer.

50 mg of nitrite degraded heparin, with alkyne functionalization at C1of the reducing terminal (prepared as in Example 3a), was dissolved in200 mL of de-ionized water and 25 mg CuSO₄×5H₂O, 50 mg sodium ascorbateand 2.9 g NaCl were added. The pH was measured to be 4.4.

The reaction between the solution of the alkyne functionalized heparinand the azide functionalized surface was carried out at room temperaturefor 1 h. Purification was performed by rinsing off non-covalently linkedheparin for 10 minutes using a 0.04 M/0.04 M borate/phosphate buffer atpH 8.0. A final rinse with de-ionized water for two minutes wasperformed to wash away buffer salt residues.

Analytical Results:

Antithrombin binding activity of bound heparin: 21 pmol/cm²

The antithrombin binding activity of bound heparin was measuredessentially as described in Pasche., et al., in “Binding of antithrombinto immobilized heparin under varying flow conditions” Artif—Organs15:481-491 (1991).

EXAMPLE 1.2 Preparation of a Non-Thrombogenic Surface on Gold

A surface comprising layers of aminated polymer and sulfatedpolysaccharide having a functionalized aminated polymer outer layer isconnected to functionalized heparin thereby forming a triazole ring.

The process of Example 1.1 was repeated with slight variation of theparameters as follows: 200 μL (2 mL/L) of a 5% solution of azidefunctionalized polyamine (prepared as in Example 2a) was employed;

The adsorption of the azide functional polyamine to the sulfate surfacewas carried out for 20 minutes at room temperature;

50 mg (250 mg/L) CuSO₄×5H₂O and 100 mg (500 mg/L) sodium ascorbate wereemployed; The pH was measured to be 4.8.

Finally, the antithrombin binding activity of bound heparin of thecoated gold surface was measured as 30 pmol/cm².

EXAMPLE 1.3 (Comparison)

Another identical gold surface was coated in the exact same way asdescribed in Example 1.2 above except that no CuSO₄ as catalyst wasadded in the heparin coupling step. The antithrombin binding activity ofbound heparin was negligible showing that if no covalent coupling occursthe heparin is rinsed off in the last buffer rinsing step.

EXAMPLE 1.4 Preparation of a Non-Thrombogenic Surface on Gold

A gold surface was coated as in Example 1.2 using native heparin, withalkyne functionalization at C1 of the reducing terminal (prepared as inExample 3b) at pH 4.8 and also at pH 7 (pH adjusted with 1 M HCl and 1 MNaOH respectively).

The antithrombin binding activity of bound heparin of the coated goldsurface was measured as 25 pmol/cm² for the surface prepared at pH 4.8and 44 pmol/cm² when the preparation was performed at pH 7.

EXAMPLE 1.5a Preparation of a Non-Thrombogenic Surface on PVC

The luminal surface of a PVC tubing (I.D. 3 mm) was cleaned withisopropanol and an oxidizing agent. It was then primed as in Example 1.1with 3 bilayers ending with sulfated polysaccharide. The priming wasthen reacted as in Example 1.2 first with an azide functionalizedpolyamine (prepared as in Example 2a) followed by reaction with nitrousacid degraded heparin, with alkyne functionalization at C1 of thereducing terminal (prepared as in Example 3a) at pH 4.8 and, separately,with native heparin, with alkyne functionalization at C1 of the reducingterminal (prepared as in Example 3b) at pH 4.8 and, separately, at pH 7.Purification was performed using the same buffer and water rinse as inExample 1.1. In both experiments, the flow used during the entireprocess was set to 100 mL/min. The antithrombin binding activity ofbound heparin for the samples was tested and found to be acceptable (i.eabove 2 pmol/cm²).

The samples were stained with toluidine blue (“TB”) (200 mg/L in water)by immersing in the solution for 2 minutes followed by extensive waterrinse. The TB attaches to the heparin via ionic interaction. As shown inFIG. 1, the stain is uniform showing that the heparin is evenlydistributed over the surface.

The coated samples were analyzed again after storage at room temperature(more than 8 months) in an aluminum foil bag with a desiccant inside toshow stability of the coating. The antithrombin binding activity ofbound heparin for the samples after aging was tested and found to beacceptable (i.e above 2 pmol/cm²).

Blood Loop Evaluation Test

Blood loop evaluation was performed on these stored samples to show thepreserved heparin bioactivity of the non-thrombogenic surface. First theluminal side of the coated tubings were washed with 0.15 M NaCl for 15hours at a flow of 1 mL/min to ensure that all loosely bound heparin wasrinsed off and a stable surface remains. Then the washed tubings wereincubated in a Chandler loop model performed essentially according toAnderson et al. (Anderson, J.; Sanchez, J.; Ekdahl, K. N.; Elgue, G.;Nilsson, B.; Larsson, R. J Biomed Mater Res A 2003, 67(2), 458-466) at20 rpm. The platelets, from fresh blood and from the blood collectedfrom the loops, were counted in a cell counter to measure the loss ofplatelets which indicates thrombosis. As references were included anon-thrombogenic control (i.e Carmeda BioActive Surface® applied to PVC,which is prepared essentially as described in EP-B-0495820), an uncoatedPVC tube, and a thrombogenic control (i.e. a three bilayer coating withan outer layer of sulfated polysaccharide not binding antithrombin).

As seen in the table below there is virtually no platelet loss (plateletloss indicates thrombosis) seen for the coatings prepared using thestored degraded and native heparin coatings prepared in this example(Example 1.5a). The uncoated PVC tubing and the surface with an outerlayer of sulfated polysaccharides (not binding antithrombin) showsignificant thrombosis in this experiment.

Platelet Loss in count × platelet Evaluated surfaces 10⁹/L count %Initial value, blood before 206 Chandler loop Evaluated surfacesDegraded heparin 203 1 according to the invention Native heparin 190 8Reference surfaces Non-thrombogenic 195 5 control Uncoated PVC tube 11644 Thrombogenic control 3 99

These results demonstrate the non-thrombogenic properties of the stablesurface prepared according to the invention.

EXAMPLE 1.5b (Comparison)

A variation on the process described in Example 1.5a was performed butnow using a polyamine that was not functionalized with azides and anitrous acid degraded heparin without an alkyne. The sample was stainedwith TB and also with Ponceau S (PS) using the procedure describedabove. The PS water solution contained 200 mg/L of PS and 5.75 mL/L ofacetic acid. As shown in FIG. 2 (upper panel) no TB stain was seen afterthis alternative procedure. However, in FIG. 2 (lower panel) a red stainfrom the PS is seen which indicates the occurrence of an outer aminatedlayer. This shows that no heparin was attached after the alternativeprocedure and that all non-covalently linked heparin is washed offduring the buffer rinse.

EXAMPLE 1.6 Preparation of a Non-Thrombogenic Surface on VariousDifferent Substrates

Different substrates (FEP, PTFE, silicone polymer, polyurethane (PU),stainless steel, and titanium) were cleaned with isopropanol and anoxidizing agent. They were then primed as in Example 1.1 with 4 bilayersending with sulfated polysaccharide. The priming was then reacted as inExample 1.2 first with an azide functionalized polyamine (prepared as inExample 2a) followed by reaction with nitrous acid degraded heparin,with alkyne functionalization at C1 of the reducing terminal (preparedas in Example 3a) at pH 7 (adjusted with 1 M HCl and 1 M NaOH).Purification was performed using the same buffer and water rinse as inExample 1.1. The coating was performed by immersing the materials intothe coating solutions.

As can be seen from FIG. 3, staining with TB (as described in Example1.5a) shows a uniform distribution of heparin on all the substrates (thesmall unstained spots are due to the fixturing of the materials).

The antithrombin binding activity of bound heparin for the differentcoated substrates are shown in the table below measured aftersterilization by ethylene oxide (EO). The EO-sterilization was performedusing a standard sterilization technique used for medical devices.

AT-uptake after EO-sterilization Substrate (pmol/cm²) FEP 19 PTFE 7.3Titanium 12 Steel 13 Silicone 7.6 PU 14

The data show that in spite of exposure to rigorous sterilizationconditions the retained activity is still acceptable.

EXAMPLE 1.7a Preparation of a Non-Thrombogenic Surface on a MedicalDevice

The luminal side of a Gore-Tex Vascular graft (thin wall, 5 mm diameter,catalog number: VT05070L) was cleaned with isopropanol. It was thenprimed as in Example 1.1 with 2 bilayers ending with sulfatedpolysaccharide. The priming was then reacted as in Example 1.2 firstwith an azide functionalized polyamine (prepared as in Example 2a)followed by reaction with nitrous acid degraded heparin, with alkynefunctionalization at C1 of the reducing terminal (prepared as in Example3a) at pH 7 (adjusted with 1 M HCl and 1 M NaOH). Purification wasperformed using the same buffer and water rinse as in Example 1.1. Theflow used during the entire process was set to 30 mL/min. The heparinantithrombin uptake activity after EO-sterilization (conditions as inexample 1.6) was measured as 8.7 pmol/cm².

The data show that in spite of exposure to rigorous sterilizationconditions the retained activity is still acceptable.

EXAMPLE 1.7b Preparation of a Non-Thrombogenic Surface on a MedicalDevice

The method of Example 1.7a may be repeated using native heparin,modified with alkyne functionalization of the reducing terminal(prepared as in Example 3b or 3c) to give a graft coated with anon-thrombogenic surface comprising modified native heparin.

EXAMPLE 1.8 Preparation of a Biocompatible Surface on a HDPE (HighDensity Poly Ethylene)

An HDPE sheet (30 cm², USP reference standard) was cleaned by immersioninto a solution of concentrated KMnO₄ (2 g/L) in concentrated H₂SO₄ for2 minutes according to the method of EP 0086186 (Larm et al). The sheetwas then primed as in Example 1.1 with 3 bilayers ending with sulfatedpolysaccharide. The priming was then reacted as in Example 1.2 firstwith an azide functionalized polyamine (prepared as in Example 2a)followed by reaction with nitrous acid degraded heparin, with alkynefunctionalization at C1 of the reducing terminal (prepared as in Example3a) at pH 7 (adjusted with 1 M HCl and 1 M NaOH). Purification wasperformed using the same buffer and water rinse as in Example 1.1. Thecoating was performed by immersing the materials into the coatingsolutions. The coating was found to be non-toxic in a cytotoxicitytesting using the Minimal Essential Medium (MEM) elution test asdescribed in ISO10993.

These results demonstrate the biocompatibility of the evaluated surface.

EXAMPLE 1.9 Preparation of a Biocompatible Surface on PVC (ReversedFunctionality and PEG Spacer)

The luminal surface of a PVC tubing (internal diameter 3 mm) was cleanedwith isopropanol and an oxidizing agent. It was then primed with fourbilayers of a positively charged polyamine (Polymin) and a negativelycharged sulfated polysaccharide (dextran sulfate) ending with thesulfated polysaccharide.

Then next coating step used a solution of 2 mL of a 5% solution ofalkyne functionalized polyamine (prepared as in Example 2b) in 1000 mLof a 0.04 M/0.04 M borate/phosphate buffer at pH 8.0. The adsorption ofthe azide functional polyamine to the sulfate surface was carried outfor 15 minutes at room temperature. A two minute water rinse wasperformed after the adsorption to rinse off excess polymer.

Then a solution of 250 mg of heparin, with azide functionalization and apolyethylene glycol (PEG) spacer at C1 of the reducing terminal, 250 mgCuSO₄×5H₂O, 50 mg sodium ascorbate, and 2.9 g NaCl in 1000 mL ofdeionized water was used. The pH was adjusted to 7 (adjusted with 1 MHCl and 1 M NaOH).

EXMPLE 1.9a

used nitrous acid degraded heparin with an azide functional group and ashort PEG spacer prepared according to Example 4a.

EXAMPLE 1.9b

used nitrous acid degraded heparin with an azide functional group and along PEG spacer prepared according to Example 4b.

EXAMPLE 1.9c

used native heparin with an azide functional group and a short PEGspacer prepared according to Example 4c.

EXAMPLE 1.9d

used native heparin with an azide functional group and a long PEG spacerprepared according to Example 4d.

The reaction between the solution with the azide functionalized heparinand the alkyne functionalized surface was carried out at roomtemperature for 1 h. Purification was performed by rinsing offnon-covalently linked heparin for 10 minutes using a 0.04 M/0.04 Mborate/phosphate buffer at pH 8.0. A final rinse with de-ionized waterfor two minutes was performed to wash away buffer salt residues.

EXAMPLE 2a Azide Functionalization of Polymin SN

Polymin SN (Lupasol SN; Lupasol is an alternative trade name forPolymin) was diluted with water to prepare a stock solution (5 g PolyminSN was added to 20 mL purified water). (Polymin is a polyethyleneiminecationic tenside available from BASF).

A solution of N-hydroxysuccinimide-azidobutyrate (Ref: Khoukhi;Vaultier; Carrie,—Synthesis and reactivity of methyl [gamma]-azidobutyrates and ethyl [sigma]-azido valerates and of the correspondingacid chlorides as useful reagents for the aminoalkylation. Tetrahedron1987, 43, (8), 1811-1822. and Malkoch, M.; Schleicher, K.;Drockenmuller, E.; Hawker, C. J.; Russell, T. P.; Wu, P.; Fokin, V. V.,Structurally Diverse Dendritic Libraries: A Highly EfficientFunctionalization Approach Using Click Chemistry. Macromolecules 2005,38, (9), 3663-3678. (see also R. Kumar, A. El-Sagheer, J. Tumpane, P.Lincoln, L. M. Wilhelmsson, T. Brown, Journal of the American ChemicalSociety, 2007, 129(21) 6859-6864) (1.7 g, 7.5 mmol) in 10 mL of purifiedwater was mixed with 24 mL of the Polymin SN (resulting in ˜5 mmol ofprimary amines in the aqueous solution) and left to react overnight at70° C. The reaction mixture was then diluted with water and isopropanol(min 99%, PhEur quality, Merck) until the polymer precipitated. Theisopropanol was decanted off and the residual isopropanol of theresulting slurry was evaporated off. The functionalized polymer wasanalyzed by NMR and FTIR. The FTIR showed a typical signal from —N₃ at2100 cm⁻¹.

EXAMPLE 2b Alkyne Functionalization of Polymin SN

Alkyne functional polyamine was prepared essentially as in Example 2abut using N-hydroxysuccinimide-(4-pentynoate) (Ref: Salmain, M.;Vessieres, A.; Butler, I. S.; Jaouen, G. Bioconjugate Chemistry 1991,2(1), 13-15) instead of N-hydroxysuccinimide-azidobutyrate.

EXAMPLE 3a Preparation of Alkyne Functionalized Nitrous Acid DegradedHeparin

Reagents:

-   -   (i) Nitrous acid degraded heparin with aldehyde groups (prepared        essentially as in Example 2 of U.S. Pat. No. 4,613,665) 3.25 g        dry weight (0.65 mmol)    -   (ii) O-(prop-2-ynyl)-hydroxylamine hydrochloride (Ref: Xu, R.;        Sim, M. K.; Go, M. L., Synthesis and pharmacological        characterization of O-alkynyloximes of tropinone and        N-methylpiperidinone as muscarinic agonists. J Med Chem 1998,        41, (17), 3220-3231) 0.70g dry weight (6.5 mmol)    -   (iii) Acetic acid (100% Merck) 3 mL    -   (iv) Purified water 50 mL

The compounds were dissolved in the mixed solvents and the pH adjustedto 4.5 with 4M NaOH. The reaction was continued for 3 days at roomtemperature. The resulting product was dialyzed against purified waterwith a SpectraPor dialysis membrane mwco 1 kD (flat width 45 mm).

The functionalized product was analyzed by FTIR which showed a typicalsignal from the alkyne at 3100 cm⁻¹.

The activity of the functionalized heparin was 96 IU/mg which indicatesthat the activity of the functionalized heparin is substantiallyunaffected by functionalization.

EXAMPLE 3b Preparation of Alkyne Functionalized Native Heparin

The native heparin (SPL, Scientific Protein Laboratories, lot no. 1037)was functionalized according to the procedures described in Example 3a.

The activity of the functionalized heparin was 211 IU/mg which indicatesthat the activity of the functionalized heparin is substantiallyunaffected by functionalization.

EXAMPLE 3c Preparation of Alkyne Functionalized Native Heparin withAromatic Spacer

The native heparin (SPL, Scientific Protein Laboratories, lot no. 1037)(20 mg) was dissolved in 250 μl acetic acid (100% Merck) and 250 μlpurified water and 6 μl N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamidefrom stock solution (see Example 5 below) was added. The reaction wascarried out at room temperature for 16 hrs. The reaction products wereconcentrated and co-evaporated with toluene (3×2 mL) to give a yellowishsolid (˜20 mg).

EXAMPLE 4 Preparation of Azide Functionalized Nitrous Acid DegradedHeparin and Native Heparin with PEG Chain Link

Preparation of Intermediates N-hydroxysuccinimidyl 4-azidobutyrate

The 4-azidobutyric acid derivative was prepared according to publishedprocedures (N. Khoukhi, M. Vaultier, R. Carrie, Tetrahedron, 1987, 43(8)1811-1822) followed by N-hydroxysuccinimide activation (R. Kumar, A.El-Sagheer, J. Tumpane, P. Lincoln, L. M. Wilhelmsson, T. Brown, Journalof the American Chemical Society, 2007, 129(21) 6859-6864).

ω-Azido Bifunctionalized Long PEG-Spacer (x˜40, See Above)

To a solution of diamino functionalized PEG (O,O′-Bis(2-aminopropyl)polypropylene glycol-block-polyethylene glycol-block-polypropyleneglycol 1900) (7.2 g; ˜3.8 mmol; x˜40) in 15 mL dichloromethane (DCM) wasadded N-hydroxysuccinimidyl 4-azidobutyrate (2.0 g, ˜8.85 mmol). Thereaction mixture was stirred at room temperature overnight, then dilutedwith DCM, and subsequently washed with 1 M HCl, NaHCO₃ (sat.) and brine.Drying (MgSO₄), concentration and drying under vacuum produced approx. 8g of a slightly yellow solid. Identification of the reaction productwith TLC and MALDI showed expected results.

ω-Azido Bifunctionalized Short PEG-Spacer (x˜11, see above)

To a solution of diamino functionalized PEG (O,O′-Bis(2-aminopropyl)polypropylene glycol-block-polyethylene glycol-block-polypropyleneglycol 500) (2.4 g; ˜3.9 mmol; x˜11) in 10 mL dichloromethane (DCM) wasadded N-hydroxysuccinimidyl 4-azidobutyrate (2.0 g, ˜8.85 mmol). Thereaction mixture was stirred at room temperature over night then dilutedwith DCM, and subsequently washed with 1 M HCl, NaHCO₃ (sat.) and brine.Drying (MgSO₄), concentration and drying under vacuum produced approx.3.1 g of an oily product. Identification of the reaction product withTLC and MALDI showed expected results.

Preparation of Azido Functionalized Heparin with Spacer (ReversedFunctionality) EXAMPLE 4a

The ω-azido bifunctionalised short PEG (800 mg, ˜1.0 mmol) was dissolvedin deionized water (35 mL), then alkyne functionalized nitrous aciddegraded heparin (500 mg, ˜0.1 mmol), see Example 3a, was added togetherwith CuSO₄×5H₂O (100 mg) and sodium ascorbate (160 mg). The reactionmixture was then stirred for 2 days followed by dialysis for 3 daysagainst purified water with a SpectraPor dialysis membrane mwco 1 kD(flat width 45 mm length 50 cm). The dialyzed product in approximately200 mL of water was filtered over a 20 um filter plate and freeze driedto yield 620 mg. The activity of the azide functionalized nitrous aciddegraded heparin with short PEG spacer was 96 IU/mg (calculated based onthe carbohydrate part) which indicates that the activity of thefunctionalized heparin is substantially unaffected by functionalization.

EXAMPLE 4b

The ω-azido bifunctionalised long PEG (2.0 g, ˜1.0 mmol) was dissolvedin deionized water (20 mL), then alkyne functionalized nitrous aciddegraded heparin (500 mg, ˜0.1 mmol), see Example 3a. was added togetherwith CuSO₄×5H₂O (100 mg) and sodium ascorbate (160 mg). The reactionmixture was then stirred for 2 days followed by dialysis for 3 daysagainst purified water with a SpectraPor dialysis membrane mwco 1 kD(flat width 45 mm length 50 cm). The dialyzed product in approximately600 mL of water was freeze dried to yield 1.8 g. The activity of theazide functionalized nitrous acid degraded heparin with long PEG spacerwas 93 IU/mg (calculated based on the carbohydrate part) which indicatesthat the activity of the functionalized heparin is substantiallyunaffected by functionalization.

EXAMPLE 4c

The ω-azido bifunctionalised short PEG (800 mg, ˜1.0 mmol) was dissolvedin deionized water (35 mL), then alkyne functionalized native heparin(1.0 g, ˜0.1 mmol), see Example 3b, was added together with CuSO₄×5H₂O(100 mg) and sodium ascorbate (160 mg). The reaction mixture was thenstirred for 2 days followed by dialysis for 3 days against purifiedwater with a SpectraPor dialysis membrane mwco 1 kD (flat width 45 mmlength 50 cm). The dialyzed product in approximately 200 mL of water wasfiltered over a 20 μm filter plate and freeze dried to yield 900 mg. Theactivity of the azide functionalized native heparin with short PEGspacer was not measured.

EXAMPLE 4d

The ω-azido bifunctionalised long PEG (1.0 g, ˜0.5 mmol) was dissolvedin deionized water (15 mL), then alkyne functionalized native heparin(450 mg, ˜0.05 mmol), see Example 3b, was added together with CuSO₄×5H₂O(50 mg) and sodium ascorbate (80 mg). The reaction mixture was thenstirred for 2 days followed by dialysis for 3 days against purifiedwater with a SpectraPor dialysis membrane mwco 1 kD (flat width 45 mmlength 40 cm). The dialyzed product in approximately 100 mL of water wasfreeze dried to yield 840 mg. The activity of the azide functionalizednative heparin with long PEG spacer was 181 IU/mg (calculated based onthe carbohydrate part) which indicates that the activity of thefunctionalized heparin is substantially unaffected by functionalization.

EXAMPLE 5 Bifunctional Linker 5 a)N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide

N-hydroxysuccinimide-(4-pentynoate) (Ref: Malkoch, M.; Schleicher, K.;Drockenmuller, E.; Hawker, C. J.; Russell, T. P.; Wu, P.; Fokin, V. V.,Structurally Diverse Dendritic Libraries: A Highly EfficientFunctionalization Approach Using Click Chemistry. Macromolecules 2005,38, (9), 3663-3678.) (200 mg, 1.0 mmol) and p-aminophenylethanol (125mg, 0.9 mmol) were dissolved in 2 mL of dichloromethane together withtriethyl amine (140 μL, 1.0 mmol), and 5 drops of dimethyl formamide.The reaction mixture was stirred at room temperature for 2 hours. Thecrude reaction product was concentrated, dissolved in 10 mL of ethylacetate and washed with 5 mL of water followed by, 5 mL of 0.5 M HCl(aq.), 5 mL of 10 NaHCO₃ (aq.) and finally 5 mL of water. The organicphase was dried with MgSO₄, filtered, and the solvent was evaporated.The product was further purified by column chromatography on silica geleluting with a gradient of toluene (T) and ethyl acetate (E) from 4:1 to1:2 (T:E). The product N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide wascharacterized by NMR and MALDI-TOF.

5 b) N-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide

N-(4-(2-(hydroxy)ethyl)phenyl)pent-4-ynamide (210 mg, 1.0 mmol) wasdissolved in 4 mL of pyridine. Methanesulfonyl chloride (MsCl) (100 μL,1.3 mmol) was added at 0° C. The stirred reaction was brought back toroom temperature and reacted at room temperature for 5 min. The solventwas evaporated and the residue re-dissolved in 10 mL of ethyl acetateand washed with 5 mL of water followed by 5 mL of 0.1 M HCl (aq.), andfinally 5 mL of water. The organic phase was dried with MgSO₄, filtered,and the solvent was evaporated to yield the productN-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide.

5 c) N-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide

The N-(4-(2-(methanesulfonate)ethyl)phenyl)pent-4-ynamide was dissolvedin 6 mL of acetonitrile and added to a solution of N-hydroxyphthalimide(200 mg, 0.9 mmol) and triethyl amine (250 μl, 1.8 mmol) in 2 mLacetonitrile. The reaction mixture was stirred at 50° C. for 2 days. Thereaction mixture was then diluted with 40 mL of ethyl acetate and washedwith 20 mL of 0.5 M HCl (aq.), 5×30 mL of 10 NaHCO₃ (aq.) to remove thered color, and finally 5 mL of water. The organic phase was dried withMgSO₄, filtered, and the solvent was evaporated. The crude product wasre-crystallized from 10 mL of toluene to obtainN-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide which wascharacterized by NMR and MALDI-TOF.

5 d) N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide

N-(4-(2-(N-oxyphthalimide)ethyl)phenyl)pent-4-ynamide (20 mg, 5.5 pmol)and ethylenediamine (200 μL, 3.0 mmol) was dissolved in 2 mL of ethanol.The reaction was stirred at 75° C. for 2 hours. The solvent wasevaporated and the crude product purified by column chromatography onsilica gel eluting with a gradient of toluene (T) and ethyl acetate(E)from 2:1 to 1:3 (T:E). The productN-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide was characterized by NMRand MALDI-TOF.

Preparation of Stock Solution:

N-(4-(2-(aminoxy)ethyl)phenyl)pent-4-ynamide (2.5 mg) was placed in ametric flask and acetonitrile (1000 μL) was added to dissolve thelinker.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications mentioned throughout thespecification of the present invention are herein incorporated in theirentirety by reference.

The invention embraces all combinations of preferred and more preferredgroups and suitable and more suitable groups and embodiments of groupsrecited above.

1-38. (canceled)
 39. A heparin moiety capable of interacting withmammalian blood to prevent coagulation or thrombus formation whichentity carries an alkyne or an azido group, which alkyne or azido groupis attached to a linker, wherein the linker is end-point attached to theheparin moiety.
 40. A heparin moiety according to claim 38 which is afull length heparin.
 41. A heparin moiety according to claim 38 whereinthe heparin moiety is covalently end-point attached through position C1of the reducing terminal to the linker.